Effective passive tumor targeting:

Regulon's nanoparticles carrying therapeutic drugs have a long circulating time in body fluids (Figure 1);

This longevity in circulation is required in finding the tumors and metastases in the body for their preferential targeting via extravasation in tumors.

This preferential targeting of cancer tissue takes advantage of the imperfections in the vasculature sprouted by tumors to accelerate their growth during neoangiogenesis; the arteries, veins and micro-vessels in normal tissue have their endothelial walls more compact compared to the "leaky" vasculature of tumors; as a result, tumors uptake 10- to 200-times more Lipoplatin nanoparticles than normal tissue (Figure 2);

This was demonstrated in human studies where patients were infused with Lipoplatin and the platinum levels were measured in surgical specimens from primary or metastatic tumors and the adjacent normal tissue.

Regulon's anticancer treatment minimizes the side effects of classic chemotherapy. In simple terms all primary tumors and metastases are being targeted regardless of the tumor type or size following intravenous administration of our drug. Their targeting depends primarily on the degree of tumor vascularization. Tumors of the stomach and breast for example have the highest degree of vascularization.

Figure 1. The scheme shows the PEGylated liposome that is the carrier of the toxic drug cisplatin with its long-circulating properties in body fluids after intravenous administration.

Figure 2. The scheme shows a blood vessel in tumor tissue. Lipoplatin nanoparticles of 100nm in diameter are depicted as spheres with the yellow toxic payload of cisplatin inside them. In normal tissue, blood vessels are impenetrable by small nanoparticles. On the contrary, tumor blood vessels have imperfections (tiny holes) in their walls (called endothelium); tumor blood vessels are established during the process of neo-angiogenesis (meaning sprouting of new blood vessels by a tumor cell mass during its growth phase). Lipoplatin nanoparticles take advantage of these tiny holes to pass through and extravasate inside the tumor reaching a concentration that can be 10- to 200-fold higher compared to the adjacent normal tissue.

Crossing of the cell membrane barrier by Lipoplatin nanoparticles leading to delivery of their toxic payload inside the cytoplasm of the tumor cell where it is needed for anticancer efficacy (Figure 3). This is a major advantage in the implementation of the treatment in the clinic to enhance efficacy and eliminate toxicity. Crossing of the cell membrane barrier by Lipoplatin was also demonstrated in cell cultures (Figure 4).Lowering of the side effects of the classical chemotherapy. Because of the lipid shell, Lipoplatin does not harm the cells of the kidney and other normal tissues to cause nephrotoxicity and other side effects. On the contrary, because of its extravasation (Figure 2) and its penetration inside the cell by fusion with the cell membrane (Figure 3) it enhances efficacy while lowering penetration into normal tissue thus lowering side effects of classical cisplatin chemotherapy (toxicity to kidneys, bone marrow, peripheral nerves, gastrointestinal tract).

Figure 3. Delivery of cisplatin “payload” directly to tumor cells facilitated by DPPG fusion circumventing the need for Ctr1-receptor mediated transportation required by naked cisplatin. After concentrating in tumors and metastases DPPG promotes the fusion of Lipoplatin with the cell membrane. Once they reach the tumor target Lipoplatin nanoparticles have the advantage, unique to Regulon’s technology, to fuse with the cell membrane of the tumor cell and empty their toxic payload inside the cytoplasm. Liposomes developed by others (e..g. Doxil of SPI-77 of Alza/J&J) are unable to do the fusion process; thus the toxic drug is emptied outside the tumor cell and is less effective.

Lipoplatin is rapidly phagocytosed by tumor cells bypassing the membrane barrier which is largely responsible for drug resistance which commonly arises with 1st line therapy. Unmodified “naked” cisplatin uses copper transporter 1 (Ctr1) receptor mediated transportation to enter tumor cells Reduced platinum uptake is a key factor in Cisplatin resistance and cisplatin resistant cancers show a lower-level of Ctr-1 expression DPPG mediated fusion and phagocytosis circumvents the need for Ctr1 transportation . Thus, Lipoplatin has the potential to treat patients that have failed previous platinum-based treatment and have developed tumors resistant to platinum drugs at the cell membrane level. An additional level of platinum drug resistance is at the level of DNA repair where resistant cells can repair DNA lesions faster.

Figure 4. Demonstration of the fusion or uptake of Lipoplatin nanoparticles using cancer cell cultures. The green donut-like structures are single cancer cells; their periphery where the cell mebrane is located fluoresces because it has uptaken fluorescent Lipoplatin nanoparticles or Regulon’s fusogenic liposomes as a control. Lipoplatin or DPPG-liposomes with fluorescent lipids enter rapidly MCF-7 human breast cancer cells thus providing proof of concept of membrane fusion or endocytosis to deliver the toxic cisplatin inside the tumor cell.

Synergy with radiation for tumor cell killing. Lipoplatin is the only nanoparticle drug available that contains a heavy metal inside a liposome. Platinum can uptake high energy from external sources such as laser or gamma rays that can burst the nanoparticle to release the toxic drug or to heat up the surrounding cytoplasm. These exciting properties are under current investigation to explore the full potential of this exciting nanoparticle.

Antiangiogenesis and antimetastasis properties. Lipoplatin nanoparticles are endowed with antiangiogenesis and antimetastasis properties. The antiangiogenesis property of Lipoplatin has been suggested from the encapsulation of the beta-galactosidase gene into a liposome of the same composition as the Lipoplatin liposome;

Figure 5. After systemic delivery to SCID mice with human tumors - the foreign “blue” gene stained preferentially the vasculature that the tumors under the skin of the animals developed to supply the tumor with nutrients.

This shows that Regulon’s liposomes can target preferentially the vascular endothelial cells; in case of Lipolatin, targeting of these cells with toxic cisplatin instead of the “blue” gene would cause their destruction. Thus, Lipoplatin limits tumor vascularization by attacking their endothelial cells in addition to the known property of cisplatin to attack the epithelial cell of the tumor.

The antimetastasis potential of Lipoplatin was shown recently at the 'Reference Oncology Center, Italian National Cancer Institute' in Aviano (http://www.ncbi.nlm.nih.gov/pubmed/24029417). Lipoplatin inhibited both migration and invasion of cervical cancer cells supporting its antimetastasis potential. This is a very important feature of Lipoplatin because migration and invasion are essential steps used by cancers to mediate their metastases.